P760: EXOSOMES FROM GRANULOCYTIC MYELOID-DERIVED SUPPRESSOR CELLS ALLEVIATE MURINE IMMUNE-MEDIATED BONE MARROW FAILURE
Authors: Ash Lee Manley, Jichun Chen, Xingmin Feng, Young Neal
Journal: HemaSphere (2023)

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Aplastic anemia (AA) is a bone marrow failure (BMF) disease characterized by marrow hypoplasia and peripheral blood pancytopenia. The majority of AA cases are immune-mediated: cytotoxic T cells destroy mature and immature hematopoietic cells. Aims: Our recent study demonstrated that granulocytic myeloid-derived suppressor cells (G-MDSCs) attenuate BMF in a minor-histocompatibility (minor-H)-mismatched AA murine model (Feng X, et al. Blood Advances. 2022). Exosomes are nanoscale, spherical, lipid bilayer vesicles secreted by cells; they can serve to deliver nucleic acids, proteins, miRNA and other bioactive substances to specific cells. The roles of exosomes have been studied extensively in various disease models. Here we extended our experiments to investigate the roles of exosomes secreted by G-MDSC in the suppression of T cell proliferation in vitro and in mitigation of murine immune AA/BMF in vivo. Methods: BM cells from C.B10-H2b/LiLMcd (C.B10) or CByB6F1 mice were incubated with Ly6G microbeads and passed through magnetic column to collect Ly6G+ G-MDSCs, which were then cultured in RPMI 1640 at 30°C for 48 hours. Exosome precipitation solution (ExoQuick-TC) was added to the collected supernatant incubated at 4°C for 24 hours and centrifuged at 3,000g for 10 minutes. Pellet was resuspended in PBS to recover exosomes, which was quantified and validated by Nano tracking particle analysis. In the T cell proliferation assay in vitro, lymph node (LN) cells from B6 mice were labeled with carboxyfluorescein succinimidyl ester (CFSE) dye and stimulated with PMA (phorbol 12-myristate 13-acetate, 50 ng/mL) and ionomycin (500 uM), and were then cultured in RPMI 1640 media at 37ºC with 5% CO2 for 5 days with or without G-MDSC exosomes. Harvested cells were analyzed by flow cytometry. In the exosome therapy study in vivo, 5 × 106 LN cells from B6 donors were infused into pre-irradiated (5 Gys) C.B10 (minor-H-mismatched) or CByB6F1 [major histocompatibility (MHC)-mismatched] recipients to induce BMF. Mice were left untreated (BMF) or were injected with exosomes originated from 20 × 106 G-MDSC at day 3 after LN cell infusion. Recipient mice were bled and euthanized at day 14 for hematological assessments. Results: In the T cell proliferation assay in vitro, G-MDSC exosomes suppressed proliferation of both CD4 and CD8 T cells (Figure 1A). In the minor-H in vivo model, when C.B10 BMF mice underwent G-MDSC-derived exosome treatment, we found significant increases in red blood cells (+51%, P<.01) and hemoglobin (+41%, P<0.05) relative to untreated BMF mice (Figure 1B). Blood neutrophils (+70%) and platelets (+17%) also increased but not significantly. G-MDSC exosome treatment decreased CD8% T cells insignificantly (-30%, P>0.05) in the BM, caused significant reduction (-30%, P<0.05) in residual BM cell apoptosis and significant increase in total BM cells (+64%, P<0.05; (Figure 1C). In the MHC mismatched model, we found no therapeutic effect when CByB6F1 BMF mice received similar treatment: red blood cells (-22%, P>0.05), hemoglobin (-23%, P>0.05), and BM cellularity were all decreased, while BM CD4 (+13%, P>0.05) and CD8 (+29%, P<0.05) T cell proportions both increased relative to BMF mice.Summary/Conclusion: Exosomes secreted by G-MDSCs suppressed T cell proliferation in vitro and attenuated immune BMF in vivo in minor-H mismatched, but not in MHC-mismatched BMF mouse models. G-MDSC exosomes could be a potential therapy for marrow failure, likely depending on the specific antigens recognized and the character of the pathologic immune response. Keywords: Aplastic anemia, Bone Marrow, Bone marrow failure, Treatment